Theaflavins are a group of polyphenolic compounds having general structure as shown in table 2 and are formed by the enzyme mediated reaction of tea polyphenol oxidase (PPO) (EC 1.10.3.1), found native to tea leaves and in other organs of tea plant with tea catechins (flavan-3-ols and their gallated esters), as its substrate, during black tea manufacture. The substrates for the formation of this reaction are present in maximum amount in tender shoots of tea plant comprising apical two leaves and an attached bud and in lesser quantities in all parts of tea plant and so is the enzyme PPO. (Reference may be made to Wickremsinghe, R. L. and Perera, K. P. W. C. (1972), Journal of the National Science Council, Sri Lanka; 1, 111-21). (Reference may also be made to Wickremsinghe, R. L., Roberts. G. R. and Perera, B. P. M. (1967), Tea quarterly; 38, 309-10). The primary substrate compounds are derivatives of flavan-3-ols commonly known as catechins as shown in table 1. Reference may be made to Yaminishi, Tei (1990), Development in Food Science, 25, Russell L. Rouseff, Bitterness in Foods and Beverages, Elsevier London. Chapter 9 wherein it is stated to include (−) epicatechin (EC); (−) epigallocatechin (EGC) and their gallate esters. Small amounts of (+) catechin and (+) gallocatechin are also found.
Catechins may be as high as 30% in Assamica varieties and only around 10% in Sinensis varieties. Reference may be made to Sanderson G. W. (1972), In Structural and Functional Aspects of Phytochemistry Runecleles V. C. ed. Academic Press, New York., 271-280 wherein it is reported that in black tea, the amount of theaflavins formed are partly 0.3-1.8 percent of the dry weight of black tea and thearubigins which are heterogeneous group of compounds comprise about 9-19% of black tea leaf. Reference may also be made to various reviews on the subject published in the past wherein chemistry of tea and its constituents are discussed in details. The matrix bound tea polyphenol oxidase is unique in terms of its high reactivity
towards tea substrates, repeated recyclability for ‘n’ number of times without losing any activity, non-adherance of formed product to matrix thus allaying fear of product poisoning of matrix bound enzyme system, enhanced thermal stability and total conversion of tea substrates to end product of theaflavins both from purified and non-purified tea substrates.
TABLE 1  R1R2M.W.(−)-EPICATECHINHH290(−)-EPIGALLOCATECHINOHH306(−)-EPICATECHIN GALLATEHGALLOYL GROUP442(−)-EPIGALLOCATECHINOHGALLOYL GROUP456GALLATE
TABLE 2 R1R2M.W.THEAFLAVINHH564THEAFLAVIN GALLATE-AHGALLOYL GROUP716THEAPLAVIN GAILATE-BGALLOYLH716GROUPTHEAFLAVIN DIGALLOYLGALLOYL GROUP868GALLATEGROUP
The polyphenol oxidase immobilized on derivatised acrylate based polymer resin helps in the production of theaflavins from tea substrates. Theaflavins are a group of condensed catechins produced during the processing of black tea. (Reference may be made to Goodsall Chris W., Safford Dick (Sep. 1-30, 1998), Second International Electronic Conference on Synthetic Organic Chemistry (ECSOC-2). These are responsible for the brightness, briskness of tea infusions and have the same antioxidant properties of free catechins, having pleasing potential of being used as food colorants, anticancer substance and important neutraceuticals. (Reference may be made to Miller, N. J., Castelluccio, C; Tijburg, L., Rice-Evans, C. (Aug. 19, 1996), FEBS Lett,; 392(1).,40-41). Being a natural product they may also be used as a coating coloring substance for tablets NAD or as anti rancid compounds in oils and fats and in cosmetic preparations. Theaflavins prevent cellular DNA damage by inhibiting oxidative stress by suppressing cytochrome P450 IAI in cell cultures. (Reference may be made to Feng, Q., Torii, Y; Uchida, K; Nakamura, Y., Hara, Y; and Osawa, T. J. (Jan. 2, 2002), Agri Food Chem; 50(1); 213-216). Theaflavins also inhibit tumor growth and inflammation (Reference may be made to. Dass, M; Sur, P; Gomes, A; Vedasiromoni, J. R. and Ganguly D. K. (2002), Phytother. Res., 16, S40-S44).
In addition to this, theaflavins also possess anti-clastogenic and anti-mutagenic effect (Reference may be made to Gupta S, Chaudhuri T, Seth P, Ganguly D. K. and Giri A. K. (2002), Phytother. Res., 16, 655-661. However, theaflavins constitute only 1.5 to 2.5 percent (dry wt.) of the black tea even though the green leaf has upto 20 percent (dry wt.) catechins. (Reference may be made to Harold, N and Graham, P. D. (1992), Green tea composition, Consumption, and Polyphenol Chemistry. Preventive Med., 21, 334-350).
The enzyme polyphenol oxidase involved in generation of theaflavins present in tea shoots (Reference may be made to Bajaj, K. L., Anan, T; Tsushida, T & Ikegaya K. (1987), J. Agric. Biol. Chem., 51, 1767-1772). has been solubilised and immobilized on the above-mentioned matrix.
Further improvements offered by the invention include the ability to maximize the biological activity retention and/or to increase the activity of target molecules, minimize the toxicity of product, minimize the reaction time at physiological pH, reduces contamination of the product and improves the stability of the activated polymer.
Limitations of other methods for immobilizing biologically active macromolecules are:    1. Long Coupling time    2. Unphysological pH leading to target molecules inactivation    3. Products contamination with either activated or inactivated polymer.    4. Polymer species or co-product toxic.    5. Limited use in aqueous solutions.    6. Activated polymer construct unstable.    7. Substantial loss of biological activity frequently seen with the cyanuric chloride method (Reference may be made to Abuchowski et al (1977a), Journal of Biological Chemistry, 252, 3582-3586) and carbonyldiimidazole method (Reference may be made to Beauchamp et al (1983), Analytical Biochemistry, 131, 25-33) and occasionally with phenylchloroformate (Reference may be made to Veronese et al (1985), Appl. Biochem. Biotechnol., 11, 141-152) and succinimedyl active ester methods (Reference may be made to Shadle et al; Katre et al (1987), Proc. Natl. Acad. Sci. USA, 84, 1487-1491).    8. Many methods recommend long coupling time and/or unphysiological pH, thus rendering target moieties less active or inactive (e.g. the carbonyldiimidazole, cyanuric chloride, phenylchloroformate and some succinimidyl active ester methods).    9. Some methods are unsuitable for use in aqueous solution, thus limiting the target molecules to those, which will tolerate non-aqueous solutions, (e.g. organic sulfonylhalide method using trifluoromethanesulfonyl chloride (Reference may be made to Mosbach & Nilson; Delgado et al (1990) Biotechnology and Applied Biochemistry, 12, 119-128).    10. Many methods use activated polymer species and/or produce co-products which are toxic in a wide range of beverages and which are potentially toxic in vivo if not separated from the product (e.g. Phenylchloroformate, cyanuric chloride methods.)In the current invention it has been found that most of the previously described methods are unsuitable because of the high levels of the product binding during active enzyme: polymer adduct reaction with substrates, causing matrix poisoning and partial or complete loss of biological activity and for unrecoverable product from active matrix, making it unsuitable as a material for bioreactor or repeated use.